Establishing ALCL cells with an inducible shRNA construct that specifically silences cellular NPM-ALK gene
To establish stably transfected cells with an inducible shRNA for ALK gene silencing, we utilized a human ALCL cell line (SUDHL-1) (Morgan et al., 1989
). Two gene-expressing constructs were introduced in a stepwise fashion as illustrated in . The ‘conditioned cells’ were generated to constitutively express Tetracycline Repressor (TetR) by transfecting ALCL cells with purified Lentiviral particles containing the vector pLenti6/CMV/TetR/Blasticidin. After selection for the Blasticidin-resistant ‘conditioned cells’, protein expression of TetR was confirmed. A Western blot indicates the presence of a 24-kDa protein band consistent with TetR in ‘conditioned cells’ (), which is not present in the parental cells. The inducible short hairpin RNA (shRNA) construct containing sequences that specifically target the C-terminal ALK portion of the NPM-ALK fusion gene were designed (shRNA-ALK), and the corresponding DNA inserts were synthesized as described in ‘Materials and Methods’. The synthesized dsDNAs encoding shRNA-ALK were cloned into a pLenti4/H1/TO/Zeocin vector and introduced into ‘conditioned cells’. To generate control cells, a non-related inducible shRNA for the Lamin gene (pLenti4/H1/TO/Zeocin/Lamin; Invitrogen) was used. Zeocin-resistant cells were examined for the presence of DNA inserts using PCR analysis of cellular DNA. As shown in the upper panel of , DNA inserts corresponding to shRNA-ALK (213-bp band in lane 5) and shRNA-Lamin (201-bp band in lane 6) were detected. No amplified DNA products were seen in the parental cells (lane 3) or in ‘conditioned cells’ (lane 4). The presence of the TetR gene in the same cells was confirmed by PCR with amplification of a 973-bp DNA product (lower panel of ). TetR was detected in ‘conditioned cells’ (lane 4), as well as in the generated cells containing the shRNA-ALK construct (lane 5) or shRNA-Lamin construct (lane 6), but it was not present in the parental cells (lane 3).
Establishing a stable ALCL cell model that contains an inducible shRNA construct for specifically silencing the ALK gene. A, Overview of cell model
To induce shRNA expression and silence the cellular ALK gene, the generated cells were treated with tetracycline (3 µg/ml) for 6 days. Western blot assays indicated that the protein expression of NPM-ALK was significantly reduced in tetracycline treated cells containing the inducible shRNA-ALK construct (, lanes 1 and 2), but was not altered in control cells carrying the inducible shRNA-Lamin construct (lanes 3 and 4).
Silencing the cellular ALK gene results in growth arrest, apoptosis, and death of ALCL cells
To obtain optimal ALK gene silencing conditions, the cells were treated with different concentrations of tetracycline for 6 days. Western blot assays indicated that the level of ALK gene silencing was tetracycline dose-dependent and reached maximal inhibition in the presence of 5 µg/ml tetracycline (). No change in NPM-ALK protein expression was observed in the control cells containing the inducible shRNA-Lamin construct. To evaluate the corresponding cellular effects that resulted from ALK gene silencing, the treated cells were simultaneously stained with trypan blue and viable cells were counted. A tetracycline dose-dependent growth inhibition was observed in cells containing the inducible shRNA-ALK construct (, p
< .01), which was not present in the control cells with inducible shRNA-Lamin. As NPM-ALK fusion proteins have a relative long half-life time of over 48 hours (Ritter et al., 2003
), a time course of ALK gene silencing was performed by treating cells with tetracycline (3 µg/ml) for 8 days. Western blot assays demonstrated that cellular NPM-ALK protein levels were significantly decreased after 4 days of tetracycline treatment, and nearly abolished after 6 days (). In addition, simultaneous studies on cell growth were carried out by counting the number of viable cells present at each time-point. Significant inhibition in the number of viable cells was seen in cells containing the inducible shRNA-ALK construct after tetracycline treatment for 6 days (, p
< .01), a timeline that closely matches the observed changes in cellular NPM-ALK protein expression. In contrast, tetracycline treatment had no effect on the growth rate of the control cells carrying inducible shRNA-Lamin.
ALK gene silencing by inducing shRNA-ALK results in cell growth arrest, apoptosis, and death. A, Dose-dependent effect of tetracycline treatment on NPM-ALK expression
To study the effect of ALK gene silencing on cell apoptosis, the generated cells were treated with tetracycline (3 µg/ml) for 4 days and stained with FITC-conjugated Annexin V. Flow cytometry analysis illustrated that silencing of the ALK gene markedly stimulated cell apoptosis from a basal level of 4.7% (, open bar) to 10.4% (closed bar, p < .01). In contrast, no change in cell apoptosis was seen in control cells carrying inducible shRNA-Lamin. Taken together, our results confirm that NPM-ALK protein expression is indispensible for ALCL cell growth and survival, and the generated cells are a useful model for studying ALK gene silencing.
Combining ALK gene silencing and the kinase inhibitor U0126 leads to synergistic inhibition of cellular JunB protein expression and ALCL cell growth
To study ALK-regulated cellular signaling pathways (Bai et al., 2000
; Zamo et al., 2002
; Chiarle et al., 2005
; Amin et al., 2007
; ), the generated cells were treated with tetracycline to induce gene silencing for 6 days as described above. Resultant changes in the activity of cellular ERK1/2, STAT3, and Akt were examined by Western blot using antibodies that recognize their phosphorylated/activated forms (p-ERK1/2, p-STAT3, and p-Akt). In addition, changes in protein expression levels of cellular JunB were studied. Simultaneously, the status of ALK gene silencing was followed by monitoring expression levels of NPM-ALK protein. As an internal control for the Western blot studies, equivalent protein loading β-actin was examined. Silencing of the ALK gene by shRNA induction with tetracycline resulted in a marked decrease of cellular p-ERK1/2, p-STAT3, p-Akt, and JunB (, lanes 1 and 2). In contrast, tetracycline treatment had no effect on control cells with inducible shRNA-Lamin (lanes 5, and 6). The abnormal expression of JunB protein mediated by CD30 activation in ALCL cells has been suggested to act via the ERK1/2 pathway (Watanabe et al., 2005
). To block the ERK1/2 pathway, cells were exposed for 2 days to the kinase inhibitor U0126 which is specific for MEK1/2, an upstream kinase of ERK1/2 (Favata et al., 1998
). As expected, exposure to U0126 (10 µM) completely inhibited ERK1/2 phosphorylation/activity and resulted in a significant decrease of JunB protein expression, while having no effect on p-STAT3, p-Akt, and NPM-ALK (lanes 3 and 7). Thus, silencing of the ALK gene or down-regulating the ERK1/2 pathway by U0126 resulted in a decrease of cellular JunB protein expression in ALCL cells. We then examined whether altering both signaling pathways (ALK and ERK1/2) would have synergistic effects. To this end, cells were treated with tetracycline for 6 days to induce ALK gene silence and were also exposed to U0126 during the last 2 days of tetracycline treatment. Changes in the activity of individual signaling pathways were examined as described above. Inhibition of both ALK gene expression (by the induced shRNA-ALK) and the ERK1/2 pathway (by addition of U0126) led to an augmented suppression of cellular JunB protein expression, but did not alter the level of p-STAT3 or p-Akt (lane 4). Furthermore, the enhanced inhibition on JunB protein expression was not seen in the control cells with inducible shRNA-Lamin (lanes 5–8). These findings suggest that although the activity of ERK1/2 pathway is partially dependent on ALK fusion protein expression in ALCL cells, it appears the ALK and ERK1/2 signaling pathways may cooperate, and independent inhibition of both pathways leads to synergistic effects on JunB protein expression.
Combining ALK gene silencing and U0126 had a synergistic effect on cellular JunB protein expression and cell growth. A, Cooperative effect of ALK and EKR1/2 pathways on cellular JunB expression
Observation of the cooperative effect on cellular JunB expression suggested that the combination of treatments might also synergistically regulate other ALCL cell functions. To this end, the cells were treated with tetracycline to induce ALK gene silencing and were simultaneously exposed to increasing doses of U0126 (0.1–5 µM) for 6 days. Cell growth rates were then calculated by the ratio of viable cells in treated conditions versus viable cells in untreated controls. As shown in , silencing of the ALK gene alone with tetracycline treatment (solid bars) led to approximately a 31% reduction in cell growth (p < .01). Exposure of cells to low doses (≤ 0.5 µM) of U0126 alone (open bars) had no effect on cell growth, although the presence of 5 µM U0126 resulted in about 37% inhibition. A significantly enhanced inhibition of cell growth (~80% reduction) was achieved by combining ALK gene silencing with a low dose (0.5 µM) of U0126 (p < .01). In contrast, this synergistic inhibitory effect was not observed with the control cells carrying inducible shRNA-Lamin, although they did respond in a similar manner to a high dose of U0126 (5 µM) with an approximately 43% reduction observed in cell growth . To further dissect the observed synergistic effect, an extended time-course study (8 days) with 0.5 µM of U0126 was performed and resultant changes in cell growth rates were examined. Silencing of the ALK gene alone via tetracycline treatment resulted in cell growth inhibition, which was initially evident at day 4 [, Tet/(−)]. When the low dose of U0126 treatment was combined with ALK gene silencing a synergistic inhibition of cell growth was observed (Tet/U0126), with the effects occurring faster (evident at day 2 when used in combination vs. day 4 for ALK gene silencing alone) and being more pronounced (~80% suppression with the combination treatments vs. 30% with ALK gene silencing alone at day 6). It is notable that the presence of 0.5 µM of U0126 alone had no effect on cell growth [(−)/U0126], with growth rates being comparable to cells receiving no treatment(s) [(−)/(−)]. In addition, the control cells with inducible shRNA-Lamin had no change in their growth rate even though they underwent similar treatments.
To verify the observed synergistic effects on cell growth, we performed MTT cell proliferation assays. The cells were treated with tetracycline to induce ALK gene silencing and simultaneously were exposed to different doses of U0126 as indicated for 4 days (). As was observed with viable cell counting experiments, the MTT assays showed silencing of the ALK gene alone caused a moderate decrease in cell proliferation [, Tet/(−), p < .01]. This ALK gene silencing-induced effect was significantly augmented by U0126 at a concentration as low as 0.5 µM (Tet/U0126, p < .01). It is important to note that at this low concentration, when used alone, U0126 had no effect on cell proliferation [(−)/U0126]. Moreover, although higher concentrations of U0126 (> 2 µM) suppressed cell proliferation, this effect could be further enhanced when combined with ALK gene silencing. In the control cells with inducible shRNA-Lamin, exposure to U0126 alone showed a similar effect on cell proliferation to the pattern seen with cells containing the inducible shRNA-ALK construct [, (−)/U0126]. However, the control cells showed no change in cell proliferation with tetracycline treatment alone [Tet/(−)], and no synergistic effect with tetracycline and U0126 (Tet/U0126). Taken together, these findings demonstrate that a combination of ALK gene silencing along with a low dose of U0126 can achieve a synergistic inhibition of ALCL cell growth/proliferation.
Synergistic inhibition of cell proliferation by combining ALK gene silencing and low dose U0126
Synergistic effects of ALK gene silencing and low dose U0126 on cell apoptosis and in vitro cell colony formation
To determine if the observed changes in cell viability were due in part to an increase in apoptosis, the cells were treated with tetracycline to induce the ALK gene silencing alone or also received a low dose of U0126 (0.5 µM) for 4 days. Apoptotic cells were stained with Annexin-V and detected by flow cytometry. Silencing of the ALK gene alone induced about a 1-fold increase in the rate of cell apoptosis (, lighter gray bar). Although a low dose (0.5 µM) of U0126 alone had no effect on the cell apoptosis rate (darker gray bar), when used in combination with ALK gene silencing the cell apoptosis rate increased by 4-fold (solid bar, p < .01). In contrast, this marked increase in cell apoptosis rate was not observed in control cells carrying the inducible shRNA-Lamin construct undergoing the same treatment(s).
Synergistic effects of ALK gene silencing and low dose U0126 on cell apoptosis and in vitro cell colony formation. A, Cell apoptosis
To evaluate the potential therapeutic value of the combination treatment, we examined the effects on in vitro cell colony formation. Cells were cultured in semi-solid methylcellulose medium containing tetracycline (3 µg/ml) to induce ALK gene silencing and/or 0.5 µM U0126 for 7 days. The formed cell colonies were then counted using light microscopy. As shown in , ALK gene silencing alone induced a moderate inhibition of cell colony formation (33% decrease; lighter gray bar). This ALK gene silencing-induced inhibition was significantly enhanced when combined with U0126 treatment (> 80% decrease; solid bar, p < .01). It should be noted that U0126 alone had only a mild inhibitory effect (darker gray bar). In the control cells carrying inducible shRNA-Lamin, no inhibition of cell colony formation was observed with tetracycline treatment alone, while a similar mild inhibitory effect was observed when U0126 was given. However, no synergistic inhibition was noted in the control cells receiving a combination of tetracycline treatment and U0126. In addition, we also examined the effects of the combination treatment on the size (diameter) of formed cell colonies using a microscope equipped with ruler lens. Neither treatment alone, tetracycline to induce ALK gene silencing nor 0.5 µM U0126 to inhibit the ERK1/2 pathway, had any effect on the size of cell colonies (, lighter and darker gray bars). However, when used together the combination treatment resulted in a significant inhibition in the size of formed cell colonies in cells containing inducible shRNA-ALK (solid bar, p < .05). In contrast, this synergistic effect was not seen in the control cells carrying an inducible shRNA-Lamin.
Combination treatment of ALK gene silencing and U0126 has a synergistic effect on recovering cell growth post-treatment
To evaluate potential post-treatment effects of the combination treatment, the cells were treated with tetracycline to induce gene silencing and/or 0.5 µM U0126 to inhibit cellular ERK1/2 pathway. As illustrated in , after 8, 10, or 12 days, the cells were washed with fresh medium to remove all treatment agents and an equal number of viable cells from each testing condition were cultured in the absence of treatment(s) for an additional 6 days. Although no significant effect on recovering cell growth was seen after an 8-day treatment course (), silencing of the ALK gene alone with tetracycline treatment for 10 days resulted in a significant inhibition in cell growth post-treatment [, Tet/(−), p < .01]. This inhibition was markedly augmented by the presence of U0126 (Tet/U0126, p < .01). Moreover, this augmented suppression on growth recovery was even more apparent after a 12-day course of the combination treatment with recovering cell growth post-treatment being nearly eliminated (, Tet/U0126, p < .01). In contrast, no change in cell growth post-treatment was detected in the control cells carrying an inducible shRNA-Lamin even after a 12-day course of the combination treatment (). These findings demonstrate that a minimum of a 10-day course with the combination treatment of ALK gene silencing and 0.5 µM U0126 is necessary to trigger prolonged post-treatment cell growth inhibition and a ≥12-day course of the combination treatment may prevent ALCL cell growth recovery post-treatment.
Prolonged inhibition of cell growth by combination of ALK gene silencing and low dose U0126. A, Overview of analysis
Combination treatment of ALK gene silencing and U0126 has a synergistic effect on ALCL tumor growth in vivo
For in vivo
studies, a mouse model with xenografted tumors was established by inoculating traceable ALCL cells containing shRNA-ALK subcutaneously in the right hind limb and control cells carrying shRNA-Lamin in the left hind limb of each mouse. After confirmation of tumor development, tumor-bearing mice were treated (n=5/condition) and resultant changes in tumor mass were monitored by whole body bioluminescence scanning to detect luciferase activity in traceable ALCL tumor cells (Figure S2
). Inducing shRNA-ALK for specific gene silencing by feeding mice tetracycline resulted in a significant decrease in ALCL tumor growth [, Tet/(−), p
< .05] and treatment with U0126 alone showed a minor effect on tumor growth [(−)/U0126]. Interestingly, the combined treatment with both ALK gene silencing and U0126 resulted in a synergistic inhibition of ALCL tumor growth (Tet/U0126, p
< .05). In contrast, inducing shRNA-Lamin alone or in combination with U0126 treatment had no additional effect on control ALCL tumor growth ().
Synergistic inhibition of xenografted ALCL tumor growth by combination of ALK gene silencing and U0126 treatment